Compositions and methods for counteracting factor xa inhibition

ABSTRACT

The disclosure provides compositions and methods for counteracting the effects of direct activated Factor X (FXa) inhibitors in a subject by administering a variant of FXa.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/759,332, filed 31 Jan. 2013, the contents of which are incorporatedherein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted concurrently herewith under 37 CFR §1.821in a computer readable form (CRF) via EFS-Web as file namePC072006_SEQLIST_ST25.txt is incorporated herein by reference. Theelectronic copy of the Sequence Listing was created on 18 Dec. 2013,with a file size of 7 kilobytes.

BACKGROUND OF THE INVENTION

Pharmacological anticoagulation is the mainstay of treatment forpatients with prothrombotic conditions. For over fifty years, the onlyoral anticoagulant available was warfarin, an inhibitor of the vitamin Kepoxide reductase (VKOR) that recycles oxidized vitamin K. Warfarin hasmany drawbacks, including unpredictable pharmacokinetics thatnecessitate frequent monitoring of coagulation parameters and doseadjustment. However, in the event of emergency bleeding or the need forurgent surgery, antidotes exist that allow rapid and complete reversal.

Oral direct FXa inhibitors are emerging anticoagulants that have thepotential to simplify dosing schemes and hemostatic monitoring inpatients with prothrombotic diseases when compared to standardtreatments, such as warfarin. Although these drugs have many advantagesover warfarin, no fully efficacious reversal agent is available forthese novel anticoagulants.

The lack of a specific countermeasure to their effects, however, is acritical unmet clinical need that could limit the widespread adoption ofthese agents due to fears of unmanageable bleeding.

SUMMARY OF THE INVENTION

Applicants have addressed this critical unmet clinical need by providingcompositions and methods for counteracting the effects of directactivated Factor X (FXa) inhibitors.

According to some embodiments, the disclosure provides methods forreducing or preventing bleeding in a subject being treated with a directFactor Xa (FXa) inhibitor by administering a composition comprising aFactor Xa variant containing at least one modification includingsubstitution for the wild-type amino acid at position 16 (using thechymotrypsin numbering system) with Thr, Leu, Phe, Asp or Gly, orsubstitution for the wild-type amino acid at position 17 (using thechymotrypsin numbering system) with Leu, Ala, or Gly. In certainembodiments, treatment with a composition comprising a FXa variantresults in at least a 50% reduction in bleeding. According to certainembodiments, direct Factor Xa inhibitors include rivaroxaban orapixaban. In some embodiments, the plasma concentration of the directFXa inhibitor is a typical therapeutic amount or a supratherapeuticamount. For example, in some embodiments, the plasma concentration ofrivaroxaban can be about 500 nM, or greater, and the plasmaconcentration of apixaban can be about 250 nM, or greater. According tocertain embodiments the FXa variant contains the substitution I16L. Insome embodiments, the FXa variant is capable of countering the effect ofthe direct Factor Xa inhibitor at a plasma concentration that is atleast 100-fold lower than the plasma concentration of the Factor Xainhibitor. In certain embodiments, the composition comprising the FXavariant is administered before a planned surgery, after an injury, orafter an intentional or accidental overdose with a direct FXa inhibitor.In some embodiments, hemostasis in the subject is monitored using ahemostasis assay after a first dose with a FXa variant and, if adequatehemostasis is not attained by a predetermined time, at least one seconddose of FXa variant is administered to achieve sufficient hemostasis.According to some embodiments, the predetermined time is about 15 mins,30 mins, 45 mins or 60 mins after the first dose of FXa variant isadministered. Other times are also possible. In some other embodiments,at least a second procoagulant is administered in addition to FXavariant, including for example, a different FXa variant, factor IX,factor XIa, factor XIIa, factor VIII, factor VIIa, FEIBA or prothrombincomplex concentrate (PCC).

According to some embodiments, the disclosure provides methods forincreasing the amount of thrombin produced in response to activation ofthe extrinsic or intrinsic clotting pathway in a subject being treatedwith a direct Factor Xa (FXa) inhibitor by administering a compositioncomprising a Factor Xa variant containing at least one modificationincluding substitution for the wild-type amino acid at position 16(using the chymotrypsin numbering system) with Thr, Leu, Phe, Asp orGly, or substitution for the wild-type amino acid at position 17 (usingthe chymotrypsin numbering system) with Leu, Ala, or Gly. According tocertain embodiments, direct Factor Xa inhibitors include rivaroxaban orapixaban. In some embodiments, the plasma concentration of the directFXa inhibitor is a typical therapeutic amount or a supratherapeuticamount. For example, in some embodiments, the plasma concentration ofrivaroxaban can be about 500 nM, or greater, and the plasmaconcentration of apixaban can be about 250 nM, or greater. According tocertain embodiments the FXa variant contains the substitution I16L.According to certain embodiments, the amount of thrombin producedincreases by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, or more. In some embodiments, the FXa variant iscapable of countering the effect of the direct Factor Xa inhibitor at aplasma concentration that is at least 100-fold lower than the plasmaconcentration of the Factor Xa inhibitor. In certain embodiments, thecomposition comprising the FXa variant is administered before a plannedsurgery, after an injury, or after an intentional or accidental overdosewith a direct FXa inhibitor. In some embodiments, hemostasis in thesubject is monitored using a hemostasis assay after a first dose with aFXa variant and, if adequate hemostasis is not attained by apredetermined time, at least one second dose of FXa variant isadministered to achieve sufficient hemostasis. According to someembodiments, the predetermined time is about 15 mins, 30 mins, 45 minsor 60 mins after the first dose of FXa variant is administered. Othertimes are also possible. In some other embodiments, at least a secondprocoagulant is administered in addition to FXa variant, including forexample, a different FXa variant, factor IX, factor XIa, factor XIIa,factor VIII, factor VIIa, FEIBA or prothrombin complex concentrate(PCC).

According to some embodiments, the disclosure provides methods fordecreasing clotting time (as measured, for example, using PT or INR, orsome other assay) in a subject being treated with a direct Factor Xa(FXa) inhibitor by administering a composition comprising a Factor Xavariant containing at least one modification including substitution forthe wild-type amino acid at position 16 (using the chymotrypsinnumbering system) with Thr, Leu, Phe, Asp or Gly, or substitution forthe wild-type amino acid at position 17 (using the chymotrypsinnumbering system) with Leu, Ala, or Gly. According to certainembodiments, direct Factor Xa inhibitors include rivaroxaban orapixaban. In some embodiments, the plasma concentration of the directFXa inhibitor is a typical therapeutic amount or a supratherapeuticamount. For example, in some embodiments, the plasma concentration ofrivaroxaban can be about 500 nM, or greater, and the plasmaconcentration of apixaban can be about 250 nM, or greater. According tocertain embodiments the FXa variant contains the substitution I16L.According to certain embodiments, clotting time is reduced by about 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In someembodiments, the FXa variant is capable of countering the effect of thedirect Factor Xa inhibitor at a plasma concentration that is at least100-fold lower than the plasma concentration of the Factor Xa inhibitor.In certain embodiments, the composition comprising the FXa variant isadministered before a planned surgery, after an injury, or after anintentional or accidental overdose with a direct FXa inhibitor. In someembodiments, hemostasis in the subject is monitored using a hemostasisassay after a first dose with a FXa variant and, if adequate hemostasisis not attained by a predetermined time, at least one second dose of FXavariant is administered to achieve sufficient hemostasis. According tosome embodiments, the predetermined time is about 15 mins, 30 mins, 45mins or 60 mins after the first dose of FXa variant is administered.Other times are also possible. In some other embodiments, at least asecond procoagulant is administered in addition to FXa variant,including for example, a different FXa variant, factor IX, factor XIa,factor XIIa, factor VIII, factor VIIa, FEIBA or prothrombin complexconcentrate (PCC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show inhibition of free wt-FXa or FXa^(I16L) by rivaroxaban.The initial velocity of peptidyl substrate (SpecXa; 200 uM) hydrolysisby A) wt-FXa (2 nM) or B) FXa^(I16L) (6 nM) was determined at increasingconcentrations of rivaroxaban. The Ki value is given on each graph.

FIGS. 2A-B show rivaroxaban inhibition of wt-FXa or FXa^(I16L) assembledin prothrombinase. The initial velocity of peptidyl substrate (SpecXa;200 uM) hydrolysis by A) wt-FXa (2 nM) or B) FXa^(I16L) (6 nM) in thepresence of PCPS (20 uM) and FVa (40 nM) was determined at increasingconcentrations of rivaroxaban.

FIG. 3 shows the effect of different concentrations of FXa^(I16L) onreversing the effects on thrombin generation of rivaroxaban.

FIGS. 4A-D show the effect of FXa^(I16L) on reversing the effects ofrivaroxaban. Normal human plasma was incubated with 500 nM rivaroxabanand in the absence or presence of increasing concentrations ofFXa^(I16L). Following data analysis, peak thrombin (A and C) and totalthrombin generated (ETP; B and D) were plotted.

FIGS. 5A-B show FXa^(I16L) reverses the effects of high doserivaroxaban. Normal human plasma was incubated with 7.5 uM rivaroxabanand in the absence or presence of increasing concentrations ofFXa^(I16L). Following data analysis, peak thrombin (A) and totalthrombin generated (ETP; B) were plotted.

FIGS. 6A-B show FXa^(I16L) or FXa^(I16T) reverses the effects of 250 nMapixaban. Normal human plasma was incubated with 250 nM apixaban and inthe absence or presence of increasing concentrations of FXa^(I16L) orFXa^(I16T). Following data analysis, peak thrombin (A) and totalthrombin generated (ETP; B) were plotted.

FIGS. 7A-B show FXa^(I16L) or FXa^(I16T) reverses the effects of highdose apixaban. Normal human plasma was incubated with 2.0 uM Apixabanand in the absence or presence of increasing concentrations ofFXa^(I16L) or FXa^(I16T). Following data analysis, peak thrombin (A) andtotal thrombin generated (ETP; B) were plotted.

FIGS. 8A-B show FXa^(I16L) corrects whole blood clotting in the presenceof rivaroxaban. Whole blood thromboelastography was used to assess theability of FXa^(I16L) to reverse the effects of rivaroxaban at a typical(A) and a high (B) dose.

FIGS. 9A-B show FXa^(I16L) corrects whole blood clotting in the presenceof apixaban. Whole blood thromboelastography was used to assess theability of FXa^(I16L) to reverse the effects of apixaban at a typical(A) and a high (B) dose.

FIGS. 10A-B show that FXa^(I16L) counteracts rivaroxaban in a thrombingeneration assay. FIG. 10A shows a dose response of rivaroxaban and FIG.10B shows a dose response of FXa^(I16L) in the presence of rivaroxaban.

FIG. 11 shows that FXa^(I16L) counteracts rivaroxaban in a mouse tailclip bleeding model.

FIG. 12 demonstrates that rivaroxaban administered to mice delaysclotting time of whole blood measured using ROTEM and thatadministration with FXa^(I16L) dose-responsively counteracts the effectof rivaroxaban.

FIG. 13 shows that rivaroxaban administered to a mouse prevents clotformation at a site of vascular injury in the cremaster muscle caused bylaser and that administration with FXa^(I16L) counteracts the effect ofrivaroxaban. Clot formation was visualized using intravital microscopyand fluorescently labeled antibodies against fibrin and platelets. FIG.13A shows clot formation in an untreated mouse. FIG. 13B shows thatrivaroxaban delayed and reduced platelet accumulation and preventedfibrin deposition. By contrast, FIG. 13C shows that in a mouseadministered rivaroxaban and FXa^(I16L) clot formation occurred at thesite of injury.

FIG. 14 is the amino acid sequence of wild-type human Factor Xpreprotein (SEQ ID NO:1). The signal peptide corresponds to amino acids1-23. The propeptide corresponds to amino acids 24-40. The mature lightchain of activated Factor X (FXa) corresponds to amino acids 41-179. Themature heavy chain of activated FXa (after removal of the activationpeptide) corresponds to amino acids 235-488. The activation peptide (AP)corresponds to amino acids 183-234.

FIG. 15 is the nucleotide sequence of the cDNA encoding wild-type humanFactor X preprotein (SEQ ID NO:2). The coding sequence corresponds tonucleotides 58 to 1524.

DETAILED DESCRIPTION

The disclosure provides compositions and methods for counteracting theanti-coagulant effect of a direct FXa inhibitor in a subject in needthereof. Applicants have discovered that certain FXa variants rapidlyand completely counteract the effect of a direct FXa inhibitor in a dosedependent manner. More specifically, applicants have discovered that arelatively small amount of an FXa variant restores normal coagulationactivity in vivo in the presence of FXa inhibitor at therapeuticconcentrations and even at supratherapeutic concentrations. By providingfast and effective antidotes for the anti-coagulation effects of directFXa inhibitors, Applicants' disclosure therefore contributes tofulfilling the promise of these advantageous anti-coagulants.

Coagulation factor X (FX) is a zymogen which, upon activation by theintrinsic factor IX/factor VIII or extrinsic pathway (tissuefactor/factor VIIa), becomes FXa, which is the protease moiety ofprothrombinase. Following proteolytic cleavage of the Arg-Ile scissilebond, releasing an activation peptide (AP), a series of well definedstructural changes in the zymogen drives the activation process to themature active serine protease (See Toso et al., (2008) J. Biol. Chem.283, 18627-18635; Bunce et al., (2011) Blood 117, 290-298; and Ivanciuet al., (2011) Nat. Biotechnol. 29, 1028-1033, incorporated by referenceherein in their entirety). The mature FXa has a light chain and a heavychain that contains the catalytic domain. The mature FXa becomes anactive serine protease upon formation of the prothrombinase complex,which includes binding of an activated cofactor, Factor Va (FVa).

Variant forms of FX have been developed that upon activation cleavageyield a zymogen-like FXa variant. That is, once cleaved, the resultingFXa variant has poor active site function and is more resistant toinactivation by circulating inhibitors (i.e. antithrombin III and TFPI).The FXa variants, thus, have longer half-lives in plasma than wild-typeFXa. The FXa variant binds FVa, lipid membrane and calcium to form afully active prothrombinase complex that efficiently activatesprothrombin.

The zymogen-like variants of FXa circulate in the zymogen-likeconformation and do not seem to be thrombogenic (See Toso et al., (2008)J. Biol. Chem. 283, 18627-18635 and Ivanciu et al., (2011) Nat.Biotechnol. 29, 1028-1033, incorporated by reference herein in theirentirety). Examples of such FXa variants are described in Internationalpatent publication WO2007/059513, incorporated herein by reference inits entirety.

The enzymes of coagulation are trypsin-like enzymes that belong to the51 peptidase family of proteases that bear a chymotrypsin-like fold. Thecoagulation proteases contain catalytic domains that are highlyhomologous to each other and to the ancestral serine proteases ofdigestion. The structural homology/identity is so great (>70%) thatresidues in the catalytic domains of the coagulation enzymes (includingFactor Xa) are numbered according to the corresponding residues inchymotrypsinogen. (Chymotrypsin numbering system; see Bajaj andBirktoft, Methods Enzymol. 1993; 222:96-128, Table 2, and Bode W, MayrI, Bauman Y, et al. The refined 1.9 Å crystal structure of humanalpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone andsignificance of the Tyr-Pro-Trp insertion segment. EMBO J 1989;8(11):3467-3475, both of which are incorporated by reference herein intheir entireties). Accordingly, amino acids may be referred to hereinaccording to the chymotrypsin numbering system, which is well-known tothose of skill in the art.

According to the disclosure, an FXa variant may be an FXa proteincomprising an amino acid substitution that makes the variant morezymogen-like compared to a wild-type FXa protein in vivo or in vitro.FXa variants of the disclosure substantially regain wild-type FXaactivity upon formation of prothrombinase. Examples of FXa variants thatare useful in methods of the disclosure are variants comprising amodification selected from the group consisting of: a) Ile at position16 is Thr, Leu, Phe, Asp or Gly and b) Val at position 17 is Leu, Ala,or Gly, according to the chymotrypsin numbering system. Amino acids 16and 17 in the chymotrypsin numbering system occur at amino acids 235 and236, respectively, of SEQ ID NO:1 (human Factor X preproprotein). Incertain embodiments, FXa variants are FXa^(I16L) and FXa^(II6T) (thenomenclature used herein for the FXa variants recites the original aminoacid at the numbered position according to the chymotrypsin numberingsystem followed by the substituted amino acid). The FXa variants can bevariants of any mammalian FXa. Of particular interest, however, are FXavariants of human FXa.

In certain embodiments, the FX variant that is activated into a variantFXa of the disclosure may be further modified by inserting a non-nativeintracellular proteolytic cleavage site. In a non-limiting example, toexpress “activated” zymogen-like FXa variants in mammalian cells, anon-native intracellular proteolytic cleavage site can be insertedbetween the Arg at position 234 of SEQ ID NO:1 (position 15 in thechymotrypsin numbering system) and the amino acid at the positioncorresponding to position 235 of SEQ ID NO:1 (position 16 in thechymotrypsin numbering system) in the variant FX zymogen. In certainembodiments, the non-native intracellular protease cleavage site isArg-Lys-Arg or Arg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO:3). These cleavagesites are efficiently recognized by proteases (PACE/furin-like enzymes)within the cell and are removed. This cleavage may result in a processedvariant FXa in which the mature heavy chain of the molecule now beginsat the amino acid at the position corresponding to position 235 of SEQID NO:1 (position 16 in the chymotrypsin numbering system). Introductionof this cleavage site at said position allows for the intracellularconversion of FX to FXa.

In certain embodiments the entire amino acid sequence of the FX variantactivation peptide (AP) (i.e., amino acids 183-234 of SEQ ID NO:1) isreplaced with a non-native intracellular protease cleavage site.According to certain embodiments, the non-native intracellular proteasecleavage site is Arg-Lys-Arg or Arg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO:3).As explained above, this modification allows for intracellular cleavageof the FX variant expressed by cells. The intracellular cleavageconverts FX variant to activated zymogen-like FXa variant which is thensecreted by cells for subsequent purification. This approach obviatesthe need for extracellular cleavage that would otherwise be required toactivate the variant clotting factor, for example, after isolating theprotein or just before blood clotting.

In certain embodiments, FXa variants of the disclosure are derived fromFX variant preproteins comprising native wild-type human signal sequenceand/or propeptide sequence. In other embodiments, FX signal sequencesand/or propeptide from non-human species can be used in place of thecorresponding native amino acid sequences. And in yet other embodiments,signal sequence and/or propeptide sequence from other human or non-humansecreted proteins can be used in place of the corresponding native aminoacid sequences.

In an exemplary embodiment, a FXa variant comprises amino acids 41-179and amino acids 235-488 of SEQ ID NO:1, wherein the amino acid atposition 235 (isoleucine in the wild-type sequence) is substituted witha different amino acid selected from the group consisting of threonine(Thr), leucine (Leu), phenylalanine (Phe), aspartic acid (Asp), orglycine (Gly). These substitutions can respectively be written using thenomenclature I235T, I235L, I235F, I235D and I235G, where the firstletter is the single letter code for isoleucine and the last letter isthe single letter code for the amino acid being substituted into thewild-type sequence. Because position 235 of SEQ ID NO:1 is equivalent toposition 16 in the chymotrypsin numbering system, the same substitutionscan be written I16T, I16L, I16F, I16D and I16G. In an embodiment, a FXavariant comprises amino acids 41-179 and amino acids 235-488 of SEQ IDNO:1, wherein the amino acid at position 235 is substituted with Thr(i.e., I235T or I16T). In an embodiment, a FXa variant comprises aminoacids 41-179 and amino acids 235-488 of SEQ ID NO:1, wherein the aminoacid at position 235 is substituted with Leu (i.e., I235L or I16L). Inan embodiment, a FXa variant comprises amino acids 41-179 and aminoacids 235-488 of SEQ ID NO:1, wherein the amino acid at position 235 issubstituted with Phe (i.e., I235F or I16F). In an embodiment, a FXavariant comprises amino acids 41-179 and amino acids 235-488 of SEQ IDNO:1, wherein the amino acid at position 235 is substituted with Asp(i.e., I235D or I16D). In an embodiment, a FXa variant comprises aminoacids 41-179 and amino acids 235-488 of SEQ ID NO:1, wherein the aminoacid at position 235 is substituted with Gly (i.e., I235G or I16G).

According to another exemplary embodiment, a FXa variant comprises aminoacids 41-179 and amino acids 235-488 of SEQ ID NO:1, wherein the aminoacid at position 236 (valine in the wild-type sequence) is substitutedwith a different amino acid selected from the group consisting ofleucine (Leu), alanine (Ala), or glycine (Gly). These substitutions canrespectively be written using the nomenclature V236L, V236A, and V236G,where the first letter is the single letter code for valine and the lastletter is the single letter code for the amino acid being substitutedinto the wild-type sequence. Because position 236 of SEQ ID NO:1 isequivalent to position 17 in the chymotrypsin numbering system, the samesubstitutions can be written V17L, V17A, and V17G. In an embodiment, aFXa variant comprises amino acids 41-179 and amino acids 235-488 of SEQID NO:1, wherein the amino acid at position 236 is substituted with Leu(i.e., V236L or V17L). In an embodiment, a FXa variant comprises aminoacids 41-179 and amino acids 235-488 of SEQ ID NO:1, wherein the aminoacid at position 236 is substituted with Ala (i.e., V236A or V17A). Inan embodiment, a FXa variant comprises amino acids 41-179 and aminoacids 235-488 of SEQ ID NO:1, wherein the amino acid at position 236 issubstituted with Gly (i.e., V236G or V17G).

In other embodiments, FXa variants of the disclosure, including thosespecific variants described in the preceding paragraphs, can includevarious isoforms of the light and/or mature heavy chain of the protein.Non-limiting exemplary isoforms of the FXa variant mature heavy chaininclude the alpha and beta versions of the mature heavy chain. Jesty etal., J Biol Chem. 1975 Jun. 25; 250(12):4497-504, incorporated byreference herein. Compositions of the disclosure can include FXa variantproteins in which one or the other or both alpha and beta mature heavychain isoforms are represented.

According to yet other embodiments, isoforms of FXa variant proteins,including those specific variants described in the preceding paragraphs,can include isoforms in which a variable number of amino acids (forexample, 1, 2, 3, 4, 5, 6, or more amino acids) are either missing fromor added to the carboxy terminus of the light chain and/or mature heavychains of the protein.

According to certain embodiments, FXa variants of the disclosure includeproteins with a certain minimal degree of homology or sequence identitycompared to the amino acid sequence of wild-type FXa in SEQ ID NO:1.Thus, for example, FXa variants include proteins that contain a lightand mature heavy chain that are at least 60%, 70%, 80%, 85%, 90%, 95%,98%, or 99% homologous or identical in sequence with the wild-type FXalight and mature heavy chains in SEQ ID NO:1, wherein such FXa variantsalso include a substitution at the amino acid position corresponding toposition 235 of SEQ ID NO:1 with Thr, Leu, Phe, Asp, or Gly, or asubstitution at the amino acid position corresponding to position 236 ofSEQ ID NO:1 with Leu, Ala, or Gly, and further wherein such FXa variantsare zymogenic until incorporated into prothrombinase complex. In theamino acid sequence of SEQ ID NO:1, the wild-type FXa light chainsequence corresponds to amino acids 41 to 179 and the wild-type FXamature heavy chain sequence corresponds to amino acids 235 to 488.Percentage amino acid sequence homology or identity can readily bedetermined using software such as Protein BLAST available at the websiteof the National Center for Biotechnology Information(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

According to other non-limiting embodiments, FXa variants of thedisclosure can also include FXa variants containing one or morepost-translational modifications including, without limitation, one ormore O-linked or N-linked carbohydrate groups or a variable number ofgamma-carboxyglutamic acid (Gla) residues. FXa variants of thedisclosure can further include chemically modified FXa variant proteins.Other FXa variants useful in the methods of the disclosure are alsopossible.

As used herein, the term FXa^(I16x) refers to a variant of activatedFactor X wherein the amino acid corresponding to position 235 in SEQ IDNO:1 (corresponding to position 16 in the chymotrypsin numbering system)is changed from the amino acid in the wild-type sequence (isoleucine) toa different amino acid denoted “x”. In some non-limiting exemplaryembodiments, amino acid “x” can be threonine (Thr or T), leucine (Leu orL), phenylalanine (Phe or F), aspartic acid (Asp or D), or glycine (Glyor G).

As used herein, the term FXa^(V17y) refers to a variant of activatedFactor X wherein the amino acid corresponding to position 236 in SEQ IDNO:1 (corresponding to position 17 in the chymotrypsin numbering system)is changed from the amino acid in the wild-type sequence (valine) to adifferent amino acid denoted “y”. In some non-limiting exemplaryembodiments, amino acid “y” can be leucine (Leu or L), alanine (Ala orA), or glycine (Gly or G).

The terms FXa^(I16x) and FXa^(V17y) are not limited by the proteinsequence set forth in SEQ ID NO:1. Rather these terms additionallyinclude the variety of isoforms and homologous proteins described hereinwith the specified substitution mutations at positions 16 or 17 in thechymotrypsin numbering system that behave as zymogens until incorporatedinto prothrombinase complex.

An FXa variant of the disclosure may be produced by any technique forexpressing a protein.

An “isolated protein,” “isolated polypeptide” or “isolated variant” is aprotein, polypeptide or variant that by virtue of its origin or sourceof derivation (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) is free of other proteinsfrom the same species, (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature. Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A protein may also be renderedsubstantially free of naturally-associated components by isolation,using protein purification techniques well known in the art.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and may be over 99% pure.Protein purity or homogeneity may be indicated by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis of aprotein sample, followed by visualizing a single polypeptide band uponstaining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The methods of the disclosure are useful to counteract a direct FXainhibitor. A direct FXa inhibitor is an inhibitor that binds directly toFXa and selectively binds FXa over other proteases. Direct FXainhibitors are noncompetitive inhibitors of FXa with respect toprothrombin. They bind the substrate binding cleft and inhibit FXacompetitively with respect to small peptide substrates that also bindthis region. They inhibit FXa with high picomolar affinity and arehighly protein bound in plasma. Examples of direct FXa inhibitors arerivaroxaban, apixaban, betrixaban, darexaban, edoxaban and otamixaban.In certain embodiments, direct FXa inhibitors are selected fromrivaroxaban and apixaban.

According to the disclosure, an FXa variant can be used to counteract adirect FXa inhibitor that binds FXa or that binds FXa that has formedprothrombinase. The direct FXa inhibitors may or may not requirecofactors of FXa for inhibition. According to the methods of thedisclosure, an FXa variant, such as FXa^(I16L) and FXa^(I16T), areadministered to a subject whose blood contains a direct FXa inhibitor.

The disclosure encompasses the use of a FXa variant to counteract directFXa inhibitors, including but not limited to synthetic inhibitors, smallmolecule inhibitors, orally available inhibitors, or reversibleinhibitors. The FXa inhibitor may be any combination of these features,such as an orally available, synthetic, reversible, small moleculeinhibitor. In certain embodiments, the direct FXa inhibitors may beselected from rivaroxaban, apixaban, betrixaban, darexaban, edoxaban andotamixaban (see Perzborn et al., Nat Rev Drug Discov. 2011 January;10(1):61-75; Turpie, Arterioscler Thromb Vasc Biol. 2007 June;27(6):1238-47; Pinto et al., Expert Opin. Ther. Patents 22:645-661(2012); Pinto, et al., J. Med. Chem. 50:5339-5356 (2007), each of whichis incorporated by reference herein). In certain embodiments, direct FXainhibitors are selected from rivaroxaban or apixaban.

In some embodiments, a FXa variant of the disclosure can be administeredto a subject to reverse the effects of a direct FXa inhibitor where suchinhibitor occurs at therapeutic concentrations. In other embodiments, aFXa variant of the disclosure can be administered to a subject toreverse the effects of a direct FXa inhibitor where such inhibitoroccurs at supratherapeutic concentrations. A supratherapeuticconcentration is one that is higher than that ordinarily consideredrequired to safely achieve anti-coagulation in a particular subject orclass of subjects. Supratherapeutic concentrations of a direct FXainhibitor can result from accidental or intentional overdose.Supratherapeutic concentrations of a direct FXa inhibitor can alsoresult from unexpected effects in particular subjects, such as anunexpectedly high sensitivity to these drugs, or unexpectedly slow rateof clearance, due for example to drug interactions or other factors.Determination of what would be a therapeutic concentration orsupratherapeutic concentration of direct FXa inhibitor in a particularsubject or class of subjects is within the knowledge of those ordinarilyskilled in the art.

According to the disclosure, an FXa variant is used to counteract adirect FXa inhibitor or inhibitors that selectively bind FXa over othertrypsin-like proteases by at least 5-fold, at least 6-fold, at least7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least25-fold, at least 30-fold, at least 50-fold, at least 100-fold, atleast, 500-fold, at least 1,000-fold, at least 5,000-fold or at least10,000-fold.

The direct FXa inhibitor may bind an FXa variant with a K_(i) of about2×10⁻⁷ M or less. “K_(i)” refers to the inhibitor constant of aparticular inhibitor-target interaction, which is the concentrationrequired to produce half maximum inhibition. One can determine the K_(i)by using methods known in the art. The disclosure contemplates, thus,counteracting a direct FXa inhibitor that binds an FXa variant free ofthe prothrombinase complex with a K_(i) of about 2×10⁻⁸ M or less, about1×10⁻⁸ M or less, about 9×10⁻⁹ M or less, about 8×10⁻⁹ M or less, about7×10⁻⁹ M or less, about 6×10⁻⁹ M or less, about 5×10⁻⁹ M or less, about4×10⁻⁹ M or less, about 3×10⁻⁹ M or less, about 2×10⁻⁹ M or less , about1×10⁻⁹ M or less, about 9×10⁻¹⁰ M or less, about 8×10⁻¹⁰ M or less,about 7×10⁻¹⁰ M or less, about 6×10⁻¹⁰ M or less, about 5×10⁻¹⁰ M orless, about 4×10⁻¹⁰ M or less, about 3×10⁻¹⁰ M or less, about 2×10⁻¹⁰ Mor less, about 1×10⁻¹⁰ M or less, about 9×10⁻¹¹ M or less, about 8×10⁻¹¹M or less, about 7×10⁻¹¹ M or less, about 6×10⁻¹¹ M or less, about5×10⁻¹¹ M or less, about 4×10⁻¹¹ M or less, about 3×10⁻¹¹ M or less,about 2×10⁻¹¹ M or less, about 1×10⁻¹¹ M or less, about 9×10⁻¹² M orless, about 8×10⁻¹² M or less, about 7×10⁻¹² M or less, about 6×10⁻¹² Mor less, about 5×10⁻¹² M or less, about 4×10⁻¹² M or less, about 3×10⁻¹²M or less, about 2×10⁻¹²M or less, or about 1×10⁻¹² M or less, or less.The direct FXa inhibitor to be counteracted by an FXa variant accordingto the methods of the disclosure may bind a wild-type FXa with a K_(i)at least 1.5 fold, at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, orat least 50-fold less than it binds the FXa variant. The direct FXainhibitor may bind a wild-type FXa with a K_(i) of at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99% less than theK_(i) with an FXa variant free of the prothrombinase complex. The directFXa inhibitor may bind a prothrombinase complex comprising a wild-typeFXa with about the same K_(i) as it binds a prothrombinase complexcomprising an FXa variant.

In one aspect, the disclosure provides methods for counteracting theeffects of a direct FXa inhibitor in a subject who is bleeding(internally or externally) or is at risk of bleeding (e.g., in thecourse of a planned surgery) by administering a FXa variant. In someembodiments, the direct FXa inhibitor may be present in the subject at atherapeutic concentration or a higher concentrations (i.e., asupratherapeutic concentration). In some embodiments, the therapeuticconcentration may be an overdose in sensitive individuals. The methodsof the disclosure, thus, are useful for providing an antidote to anoverdose of a direct FXa inhibitor. In various embodiments, the subjectof treatment may be a human or a veterinary subject.

Direct inhibitor overdose can be detected based on existence of symptomsor signs of excessively reduced clotting ability. Non-limiting examplesinclude evidence of gastrointestinal bleeding, including dark tarrystools, bloody stools, and vomiting of blood. Other examples includenosebleeds, and increased tendency to, or severity of, bruising orbleeding from minor cuts and scrapes.

In a clinical setting, direct inhibitor overdose can be detecteddirectly or by measuring the ability of subject blood to clot anddetecting deviations from the expected degree of anti-coagulation. Bloodclotting potential can be measured in ways familiar to those ordinarilyskilled in the art. For example, overdose may be suspected when asubject's prothrombin time is excessively prolonged. In certainembodiments, overdose is confirmed when the prothrombin time expressedas an International Normalized Ratio (INR) is measured to be greaterthan about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 12, 14, 16, 18, 20, or greater.

The FXa variant may be administered whenever it is desired to counteractthe effects of the direct FXa inhibitor, including but not limited tobefore a planned surgery, after an injury resulting in external orinternal bleeding or after a direct FXa inhibitor overdose. According tothe disclosure, the FXa variant may be administered at least about 12hours, at least about 6 hours, at least about 3 hours, at least about 2hours, at least about 1 hour, at least about 30 minutes, at least about10 minutes, or at least about 5 minutes of when the desiredcounteracting effect is needed, such as before a planned surgery, afteran injury resulting in external or internal bleeding or after a directFXa inhibitor overdose.

According to another embodiment, the disclosure provides a method ofadministering a FXa variant to effect the urgent reversal of acquiredcoagulopathy due to FXa inhibition therapy in a subject with acute majorbleeding. In some embodiments, subjects are adult human patients. Inother embodiments, subjects are pediatric human patients.

In some embodiments, acute major bleeding is caused by trauma. In otherembodiments, acute major bleeding occurs during surgery or other type ofinterventional procedure. Exemplary non-limiting interventionalprocedures include incisions, drainage, vascular surgery, appendectomy,herniotomy or hernioplasty, abdominal surgery, cholecystectomy,trephination (burr hole), lumbar puncture, cardiac pacemaker insertion,hip fracture surgery, and others. In yet other embodiments, acute majorbleeding can be spontaneous bleeding with no apparent cause.

Without limitation, sites of acute major bleeding includegastrointestinal bleeding, subcutaneous or intramuscular bleeding,bladder bleeding, hemarthrosis, subdural hematoma, nasal bleeding,peritoneal bleeding, uterine bleeding, and other sites of bleeding.

Effective treatment with FXa variants of the disclosure can reverse theeffects of a direct FXa inhibitor. Successful reversal of such effectsby a FXa variant can be determined in a variety of ways and be measuredor monitored using different assays, methods, or endpoints.

In some embodiments, treatment with a FXa variant to reverse the effectsof a direct FXa inhibitor is monitored using tests or assays performedon blood or plasma from a subject treated with FXa variant. A bloodsample can be taken from a subject at a predetermined time aftertreatment with FXa variant. The blood, or plasma prepared from it, isthen subjected to one or more tests to determine if certain hemostaticpharmacodynamic parameters have been normalized despite the presence ofdirect FXa inhibitor. If normalization is found then the subject neednot be further treated with FXa variant. If normalization is not found,however, then further treatment with FXa variant in accordance with themethods of the disclosure may be required to reverse the effect of adirect FXa inhibitor. Tests for monitoring the effectiveness oftreatment with a FXa variant include tests that directly or indirectlymeasure the ability to clot or that measure the activity of a direct FXainhibitor. Non-limiting exemplary tests include prothrombin time or therelated International Normalized Ratio, the prothrombinase-inducedclotting time assay, thromboelastometry, thromboelastography,chromogenic anti-FXa assay, thrombin generation assay, level ofprothrombin fragment 1+2, level of thrombin-antithrombin III complex,activated partial thromboplastin time, and partial thromboplastin time.Other tests are also possible within the knowledge of those of ordinaryskill in the art.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant reduces bleedingin the subject. In some embodiments, treatment with FXa variant reducesbleeding in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% in the presence of a direct FXa inhibitor compared toabsence of treatment with FXa variant. In other embodiments, treatmentwith FXa variant reduces bleeding in a subject about 5%-10%, 10%-15%,15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%,55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,or 95%-100%.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant reduces theactivity of a direct FXa inhibitor in the subject. In some embodiments,treatment with FXa variant reduces activity of the direct FXa inhibitorin a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 99% in the presence of a direct FXa inhibitor compared to absence oftreatment with FXa variant. In other embodiments, treatment with FXavariant reduces the activity of a direct FXa inhibitor in a subjectabout 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%,40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%,80%-85%, 85%-90%, 90%-95%, or 95%-100%.

Activity of a direct FXa inhibitor can be monitored using a chromogenicanti-FXa assay, such as that described in Asmis, et al., Thromb Res.,129:492-498 (2012), or Barrett, et al., Thromb Haemost. 104:1263-71(2010), each of which are incorporated by reference herein.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant increases theamount of thrombin produced in the blood or plasma of the subject. Insome embodiments, treatment with FXa variant increases thrombinproduction in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold,or more in the presence of a direct FXa inhibitor compared to theabsence of an FXa variant. Thrombin production in the blood or plasma ofa subject can be determined using the thrombin generation assay (TGA) orother technique familiar to those of ordinary skill in the art.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant increases clottingin the subject. In some embodiments, treatment with FXa variantincreases clotting in a subject at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least50-fold, or more in the presence of a direct FXa inhibitor compared tothe absence of an FXa variant.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant reduces clottingtime in the subject. In some embodiments, treatment with FXa variantreduces clotting time in a subject at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXainhibitor compared to absence of treatment with FXa variant. In otherembodiments, treatment with FXa variant reduces clotting time in asubject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%,35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%,75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.

According to some embodiments, clotting time is determined by measuringthe subject's prothrombin time (PT) which decreases as hemostasis isrestored. PT is the amount of time it takes for serum to clot afteraddition of tissue factor. PT therefore measures the capability of theextrinsic clotting system to support clotting. PT can vary depending onthe particular reagents a lab uses to run the test, but a normal PT isabout 11 to 13 seconds. Clotting time can also be expressed using theInternational Normalized Ratio (INR), which eliminates lab to labvariability in clotting time measurements. Using the INR, a ratio of 0.8to 1.1 indicates normal clotting. PT or INR can be determined at apredetermined time after a FXa variant is administered to a subject inneed of reversal of the effects of a direct FXa inhibitor.

In some embodiments, treatment with a FXa variant to reverse the effectsof a direct FXa inhibitor reduces the PT of a subject to about 25seconds, 24 seconds, 23 seconds, 22 seconds, 21 seconds, 20 seconds, 19seconds, 18 seconds, 17 seconds, 16 seconds, 15 seconds, 14 seconds, 13seconds, 12 seconds, 11 seconds, 10 seconds, or less. In otherembodiments, treatment with a FXa variant reduces the INR or a subjectto about 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9,2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5,1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or less. According to otherembodiments, treatment with FXa variant reduces PT or INR in a subjectabout 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%,40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%,80%-85%, 85%-90%, 90%-95%, or 95%-100%.

Prothrombin time can be measured at a predetermined after administrationof a FXa variant. Thus, in some non-limiting embodiments, PT is measured15 mins, 20 mins, 30 mins, 40 mins, 45 mins, 50 mins, 60 mins or moreafter administration of FXa. Other times are also possible according tothe knowledge of those of ordinary skill in the art.

Clotting time can also be measured using the one-stepprothrombinase-induced clotting time (PiCT) assay as described in Graff,et al., Monitoring effects of direct FXa-inhibitors with a new one-stepprothrombinase-induced clotting time (PiCT) assay: comparative in vitroinvestigation with heparin, enoxaparin, fondaparinux and DX 9065a, Int JClin Pharmacol Ther., 45:237-43 (2007) and Harder, et al., Monitoringdirect FXa-inhibitors and fondaparinux by Prothrombinase-inducedClotting Time (PiCT): relation to FXa-activity and influence of assaymodifications, Thromb Res.,123:396-403 (2008), each of which areincorporated by reference.

In yet other embodiments, the methods of thromboelastometry orthromboelastography may be used to analyze clot formation or clottingtime.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant increases thelevel of prothrombin fragment 1+2 (PF1+2) in the blood or plasma of thesubject. In some embodiments, treatment with FXa variant increases PF1+2in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold,15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in thepresence of a direct FXa inhibitor compared to the absence of an FXavariant.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant increases thelevel of thrombin-antithrombin III complex (TAT) in the blood or plasmaof the subject. In some embodiments, treatment with FXa variantincreases TAT in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold,or more in the presence of a direct FXa inhibitor compared to theabsence of an FXa variant.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant reduces activatedpartial thromboplastin time (aPTT) in the subject. In some embodiments,treatment with FXa variant reduces activated partial thromboplastin time(aPTT) in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% in the presence of a direct FXa inhibitor compared toabsence of treatment with FXa variant. In other embodiments, treatmentwith FXa variant reduces aPTT in a subject about 5%-10%, 10%-15%,15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%,55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,or 95%-100%.

According to some embodiments, reversing the effects of a direct FXainhibitor in a subject by administering a FXa variant reduces partialthromboplastin time (PTT) in the subject. In some embodiments, treatmentwith FXa variant reduces partial thromboplastin time (PTT) in a subjectat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in thepresence of a direct FXa inhibitor compared to absence of treatment withFXa variant. In other embodiments, treatment with FXa variant reducesPTT in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%,30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%,70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.

In other embodiments, clinical endpoints can be relied upon to determineif hemostasis has been adequately restored in a subject treated with aFXa variant to reverse the effects of a direct FXa inhibitor. Forexample, where a subject presents with acute bleeding, clinicalhemostatic efficacy can be scored “very good” where prompt cessation ofexisting bleeding occurs after treatment with FXa variant;“satisfactory” where there is a 1-2 hr delay in bleeding cessation;“questionable” where there is a >2 hr delay in bleeding cessation; and“none” where an effect on bleeding is absent. Where treatment with FXavariant is determined to be less than satisfactory, then an additionaldose of FXa variant can be administered to effect adequate hemostasis.In a further example, where a subject is undergoing an interventionalprocedure, clinical hemostatic efficacy can be scored “very good” wherenormal hemostasis is attained during the procedure; “satisfactory” whereintraprocedural hemostasis is mildly abnormal as judged by quantity orquality of blood loss (e.g., slight oozing); “questionable” whereintraprocedural hemostasis is moderately abnormal as judged by quantityor quality of blood loss (e.g., controllable bleeding); and “none” whereintraprocedural hemostasis is severely abnormal as judged by quantity orquality of blood loss (e.g., severe refractory hemorrhage).

A therapeutically effective dose of a direct FXa inhibitor depends uponnumerous factors that are well known to a medical practitioner of skillin the art. A typical therapeutic plasma concentration of rivaroxaban isabout 500 nM. However, according to the disclosure, an FXa variant canbe administered to counteract lower or higher concentrations ofinhibitor. The plasma concentration of rivaroxaban in a subject to betreated with an FXa variant may be lower or higher than the typicaltherapeutic concentration, for example about 100 nM, about 200 nM, about300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about800 nM, about 900 nM or about 1,000 nM.

A typical therapeutic plasma concentration of apixaban is about 250 nM.In certain embodiments, the FXa variant is administered to a subjectwith a plasma concentration of apixaban of about 100 nM, about 200 nM,about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM,about 800 nM, about 900 nM or about 1,000 nM.

Likewise, according to the disclosure, an FXa variant can be used tocounteract a direct FXa inhibitor in cases of overdose, such as when theplasma concentration of the inhibitor is at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or at least 1.5 fold, at least2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 30-fold, or at least 50-fold higherthan the typical therapeutic plasma concentration.

The FXa variants are surprisingly effective in counteracting a directFXa inhibitor at a plasma concentration that is lower than the plasmaconcentration of the direct FXa inhibitor. According to the disclosure,the FXa variant counters the effect of a direct FXa inhibitor at aplasma concentration ratio of variant to inhibitor of about 1 to 10,about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 250, about 1 to500, about 1 to 1,000, about 1 to 2,500, about 1 to 5,000 or about 1 to10,000. In certain embodiments, the FXa variant counters the effect of adirect FXa inhibitor at a plasma concentration of at least 10-fold, atleast 25-fold, at least 50-fold, at least 100-fold, at least 250-fold,at least 500-fold, at least 1,000-fold, at least 2,500-fold, at least5,000-fold, or at least 10,000-fold lower than the plasma concentrationof the direct FXa inhibitor.

In other embodiments, the plasma concentration of an FXa variantsufficient to reverse the effect of a direct FXa inhibitor is calculatedby multiplying the plasma concentration of the direct inhibitor by aconversion factor ranging from about 0.1×10⁻⁴ to about 1000×10⁻⁴, about4×10⁻⁴ to about 40×10⁻⁴, about 20×10⁻⁴ to about 200×10⁻⁴, or otherranges. In yet other embodiments, the conversion factor can be about0.1×10⁻⁴, 0.5×10⁻⁴, 1×10⁻⁴, 2×10⁻⁴, 3×10⁻⁴, 4×10⁻⁴, 5×10⁻⁴, 6×10⁻⁴,7×10⁻⁴, 8×10⁻⁴, 9×10⁻⁴, 10×10⁻⁴, 11×10⁻⁴, 12×10⁻⁴, 13×10⁻⁴, 14×10⁻⁴,15×10⁻⁴, 16×10⁻⁴, 17×10⁻⁴, 18×10⁻⁴, 19×10⁻⁴, 20×10⁻⁴, 21×10⁻⁴, 22×10⁻⁴,23×10⁻⁴, 24×10⁻⁴, 25×10⁻⁴, 26×10⁻⁴, 27×10⁻⁴, 28×10⁻⁴, 29×10⁻⁴, 30×10⁻⁴,31×10⁻⁴, 32×10⁻⁴, 33×10⁻⁴, 34×10⁻⁴, 35×10⁻⁴, 36×10⁻⁴, 37×10⁻⁴, 38×10⁻⁴,39×10⁻⁴, 40×10⁻⁴, 45×10⁻⁴, 50×10⁻⁴, 55×10⁻⁴, 60×10⁻⁴, 65×10⁻⁴, 70×10⁻⁴,75×10⁻⁴, 80×10⁻⁴, 85×10⁻⁴, 90×10⁻⁴, 95×10⁻⁴, 100×10⁻⁴, 110×10⁻⁴,120×10⁻⁴, 130×10⁻⁴, 140×10⁻⁴, 150×10⁻⁴, 160×10⁻⁴, 170×10⁻⁴, 180×10⁻⁴,190×10⁻⁴, 200×10⁻⁴, 250×10⁻⁴, 300×10⁻⁴, 350×10⁻⁴, 400×10⁻⁴, 450×10⁻⁴,500×10⁻⁴, 550×10⁻⁴, 600×10⁻⁴, 650×10⁻⁴, 700×10⁻⁴, 750×10⁻⁴, 800×10⁻⁴,850×10⁻⁴, 900×10⁻⁴, 950×10⁻⁴, or 1000×10⁻⁴, and ranges among thesenumbers. Plasma concentration of FXa direct inhibitor can be measuredaccording to the knowledge of the skilled artisan, for example, byradio-immuno assay (RIA) or other method.

Achieving a target plasma concentration of FXa variant sufficient toreverse overdose of a direct FXa inhibitor is within the knowledge ofthose ordinarily skilled in the art. In a non-limiting example,estimates of relevant pharmacokinetic parameters, such as subject plasmavolume or other parameters, can be made based on upon subject sex,height and weight, or other factors, and used to calculate how much FXavariant needs be administered to achieve the target concentration. Afteradministering FXa variant, plasma concentrations can be monitoredaccording to the knowledge of those ordinarily skilled in the art andthis information used to maintain the concentration in any desiredrange.

The compositions and methods of the disclosure include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an FXa variant. A “therapeutically effective amount” refersto an amount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the FXa variant may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the FXa variant to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the FXa variant are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. For example,a dose may be given prior to a planned surgery.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe disclosure are dictated by and directly dependent on (a) the uniquecharacteristics of the FXa variant and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an FXa variant for the treatment ofindividuals.

In certain embodiments, a therapeutically or prophylactically-effectiveamount of an FXa variant administered is about 0.0001 to 50 mg/kg, about0.001 to 50 mg/kg, about 0.001 to 5 mg/kg, about 0.001 to 0.5 mg/kg,about 0.001 to 0.05 mg/kg, about 0.01 to 5 mg/kg or about 0.01 to 0.5mg/kg.

In certain embodiments, a therapeutically or prophylactically-effectiveserum concentration of an FXa variant of the disclosure is about 0.0003to 300 nM, about 0.003 to 300 nM, about 0.03 to 300 nM, about 0.003 to30 nM, about 0.03 to 30 nM or about 0.3 to 3 nM. The concentration ofthe FXa variant, for example in blood or plasma, may be measured by anymethod known in the art.

It is to be noted that dosage values may vary with FXa inhibitorconcentration. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Another aspect of the present disclosure provides kits comprising an FXavariant or a composition comprising such an FXa variant. A kit mayinclude, in addition to the FXa variant or composition, diagnostic oradditional therapeutic agents. A kit can also include instructions foruse in a therapeutic method, as well as packaging material such as, butnot limited to, ice, dry ice, styrofoam, foam, plastic, cellophane,shrink wrap, bubble wrap, cardboard and starch peanuts. In oneembodiment, the kit includes the FXa variant or a composition comprisingit and one or more therapeutic agents that can be used in a methoddescribed herein.

The FXa variant may be administered, for example in a compositioncomprising it, once or multiple times to a subject until adequatehemostasis is restored or the direct FXa inhibitor or inhibitors are nolonger effective. Where multiple administrations are used they mayadministered hourly, daily, or at any other appropriate interval,including for example multiple daily doses. Multiple doses may beadministered on a schedule such as every 10 minutes, every 15 minutes,every 20 minutes, every 30 minutes, every hour, every two hours, everythree hours, every four hours, three times daily, twice daily, oncedaily, once every two days, once every three days, and once weekly. TheFXa variant may also be administered continuously, e.g. via a minipump.The FXa variant may be administered, for example, via a parenteral route(e.g., intravenously, subcutaneously, intraperitoneally, orintramuscularly). The FXa variant will generally be administered as partof a pharmaceutical composition as described below.

In another embodiment, the FXa variant may be co-administered withanother procoagulant including another FXa variant, Factor IX, FactorXIa, Factor XIIa, Factor VIII, Factor VIIa, FEIBA and prothrombincomplex concentrate (PCC).

Co-administration of an FXa variant of the disclosure with an additionaltherapeutic agent (combination therapy) encompasses administering apharmaceutical composition comprising the FXa variant and the additionaltherapeutic agent, as well as administering two or more separatepharmaceutical compositions, i.e., one comprising the FXa variant andthe other(s) comprising the additional therapeutic agent(s).Co-administration or combination therapy further includes administeringthe FXa variant and additional therapeutic agent(s) simultaneously orsequentially, or both. For instance, the FXa variant may be administeredonce every three days, while the additional therapeutic agent isadministered once daily at the same as the FXa variant, or at adifferent time. An FXa variant may be administered prior to orsubsequent to treatment with the additional therapeutic agent.Similarly, administration of an FXa variant of the disclosure may bepart of a treatment regimen that includes other treatment modalitiesincluding surgery. The combination therapy may be administered toprevent recurrence of the condition. The combination therapy may beadministered from multiple times hourly to weekly. The administrationsmay be on a schedule such as every 10 minutes, every 15 minutes, every20 minutes, every 30 minutes, every hour, every two hours, every threehours, every four hours, three times daily, twice daily, once daily,once every two days, once every three days, once weekly, or may beadministered continuously, e.g. via a minipump. The combination therapymay be administered, for example, via a parenteral route (e.g.,intravenously, subcutaneously, intraperitoneally, or intramuscularly).

In a further aspect, the disclosure provides a composition comprising anFXa variant for use in counteracting a direct FXa inhibitor in asubject. The composition may comprise a pharmaceutically acceptablecarrier, vehicle or other ingredients that are physiologicallycompatible. Non-limiting examples of such carriers, vehicles and otheringredients include solvents (e.g., water, ethanol, saline, phosphatebuffered saline), detergents, surfactants, dispersion media, coatings,antibacterial or antifungal agents, isotonifying agents, absorptiondelaying agents, sugars (e.g., sucrose, dextrose, lactose), polyalcohols(e.g., glycerol, mannitol, sorbitol), salts (e.g., sodium chloride,potassium chloride), wetting agents, emulsifying agents, preservatives,buffers, and agents capable of enhancing the stability or effectivenessof the FXa variant.

A composition for use according to the disclosure may be in any suitableform for administration to a subject, such as liquid solutions (e.g.,injectable and infusible solutions). Compositions can be provided in apre-mixed format ready for administration to a subject, for example, ina vial or pre-filled syringe. Such formats do not require reconstitutionwith diluent before administration. Alternatively, compositions can beprovided in lyophilized form requiring reconstitution with diluent(e.g., sterile water or saline) before administration. If the latter,diluent can be provided with the lyophilisate in a separate container.According to the knowledge of those of ordinary skill in the art,compositions can be formulated for storage under refrigeration or atroom temperature. The form of the composition depends, at least in part,on the intended mode of administration. In certain embodiments, the modeof administration is parenteral, including for example intravenous,subcutaneous, intraperitoneal, or intramuscular administration.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, in liposomes, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the FXa variant in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

It is further contemplated by the present disclosure that any of thecompositions herein may be administered to a subject being treated witha direct FXa inhibitor.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be apparent to persons skilled in the art and areto be included within the and can be made without departing from thetrue scope of the invention.

EXAMPLES Example 1 FXa^(I16L) Sensitivity Toward Rivaroxaban

To test the sensitivity of FXa^(I16L) toward rivaroxaban, inhibitionassays were established. Rivaroxaban was an efficient inhibitor ofwild-type FXa exhibiting an inhibition constant (K_(i)) of 0.582 nM(FIG. 1A). Due to the zymogen-like nature of FXa^(I16L), rivaroxabanbound with a ˜15-fold reduced affinity to this variant (K_(i)=9.3 nM)(FIG. 1B). In contrast, when the variant was assembled in theprothrombinase complex (i.e. upon addition of FVa and phospholipidvesicles), the K_(i) for rivaroxaban was nearly restored to a valuecomparable to the wild-type enzyme (wt FXa, K_(i)=2.67 nM (FIG. 2A);FXa^(I16L), K_(i)=3.4 nM (FIG. 2B).

Example 2 FXa Variants Counteract Rivaroxaban and Apixaban

The thrombin generation assay (TGA) was used to assess whether thezymogen-like FXa variants can reverse the effects of direct FXainhibitors in a more physiologic environment. The TGA measures thrombinproduction in plasma over time following the initiation of coagulationand was performed as previously described (See Bunce et al., (2011)Blood 117, 290-298, incorporated by reference herein in their entirety).Thrombin generation in normal human plasma was measured for 90 min at37° C. in the presence or absence of 500 nM rivaroxaban. To evaluate ifFXa^(I16L) can reverse the effect of rivaroxaban, increasing amounts ofFXa^(I16L) was added to plasma containing 500 nM rivaroxaban. Thrombingeneration was initiated with 2.0 pM tissue factor/4 uM phospholipid aswell as CaCl₂ and a thrombin fluorogenic substrate.

The data demonstrated that the thrombin generation profile of plasma inthe presence of 500 nM rivaroxaban was substantially reduced compared toplasma in the absence of rivaroxaban. In contrast, increasingconcentrations of FXa^(I16L) from 0.03 to 1 nM restored thrombingeneration (FIG. 3). These data show that unexpectedly lowconcentrations of FXa^(I16L) in the nanomolar and subnanomolar range canreverse the effects of the inhibitor. Dose response analysis ofFXa^(I16L) in the presence of 500 nM rivaroxaban (a typical therapeuticplasma concentration) shows that the peak height of thrombin generation(FIGS. 4A and C) and total thrombin produced (ETP) (FIGS. 4B and D)essentially reached a maximum and was completely restored to normallevels between 1-3 nM of FXa^(I16L) under these conditions. Furtherexperiments showed that even in the presence of high concentration ofrivaroxaban (7.5 μM; supratherapeutic), FXa^(I16L) was still quiteeffective at a relatively low dose 3.0 nM) in restoring peak thrombin(FIG. 5A) as well as total thrombin generated (FIG. 5B).

Similar experiments were also performed to evaluate whether FXazymogen-like variants could reverse the effects of another direct FXainhibitor, apixaban. In these experiments, the effectiveness ofFXa^(I16L) with another zymogen-like FXa variant, FXa^(I16T), was alsocompared. FXa^(I16T) is similar to FXa^(I16L), however it hasintrinsically less activity, has a longer plasma half-life, and has˜3-5-fold reduced activity compared to FXa^(I16L) when assembled in theprothrombinase complex. Consistent with the rivaroxaban data, FXa^(I16L)could restore peak thrombin (FIG. 6A) and total thrombin (FIG. 6B)generated in the presence of 250 nM apixaban (a typical therapeuticplasma concentration) in a dose-dependent manner, which appears to reacha maximum between 1-3 nM of FXa^(I16L). FXa^(I16T) was also effective atreversing the effects of apixaban; however, it appears that higherconcentrations of this variant are needed to fully restore thrombingeneration (FIG. 6). Both variants were still effective even in thepresence of a higher concentration of apixaban (2 μM). However, underthese conditions it appears that higher concentrations of both variantsare needed to fully restore thrombin generation (FIG. 7A and B).

Example 3 FXa^(I16L) Counteracts Rivaroxaban and Apixaban in Whole Blood

Whole blood thromboelastometry was used to assess the ability of theFXa^(I16L) variant to reverse the effects of the direct FXa inhibitorsin whole blood. In this system, blood is drawn from healthy volunteers.The first 2 mL of blood were discarded and the subsequent 5 mL of bloodwas collected into a vacutainer (BD, Franklin Lakes, N.J.). Corn trypsininhibitor and sodium citrate were in the collection tube, prior tocollection of the blood sample, to achieve a final concentration of0.105 M citrate and 25 μg/mL corn trypsin inhibitor (HaematologicTechnologies, Burlington, Vt.) in the blood. Two sets of reactions wereanalyzed for each donor. The initial reaction initiated 5 minutes postblood collection. The second reaction initiated 1 hour post initiationof first reaction (1 hour 5 minutes after collection).

Blood was analyzed using Thromboelastometry ROTEM®delta (TemInternational GmbH, Munich, Germany). For the reaction: (1) 6 μL ofvehicle, protein, and/or inhibitor were added to the empty cup, (2) 20μL of 0.2 M CaCl₂ (final concentration 11.6 mM), and (3) 20 μL ofInnovin (final concentration in reaction 1:10,000; source of tissuefactor) were added to the cup. Whole blood collected as described abovewas added to the reaction (300 μL) and recordings were allowed toproceed for approximately 30-60 minutes. The data collected was analyzedusing the manufacture's software (Rotem Gamma Software Version 1.1.1).

The ability of FXa^(I16L) to accelerate whole blood clot formation inthe presence of rivaroxaban or apixaban was examined using rotationalthromboelastometry (ROTEM). Both direct FXa inhibitors alone and at twodifferent concentrations have a substantial effect on whole blood clotformation: at low doses (therapeutic concentrations), whole blood clotformation is partially eliminated (FIG. 8A and FIG. 9A), while at highdoses (supratherapeutic concentrations), whole blood clot formation isalmost completely eliminated (FIG. 8B and FIG. 9B). The effects ofeither rivaroxaban or apixaban on whole blood clot formation could bereversed by FXa^(I16L). In the presence of either 500 nM rivaroxaban or250 nM apixaban, 0.3 nM FXa^(I16L) could fully or nearly fully restorewhole blood coagulation (FIG. 8A and FIG. 9A). When higherconcentrations of the direct FXa inhibitors were used (˜2 uM), 0.3 nMFXa^(I16L) partially restored whole blood coagulation and 3 nMFXa^(I16L) fully restored it (FIG. 8B and FIG. 9B). These datademonstrated that an FXa zymogen-like variant can effectively reversethe anticoagulant effect of rivaroxaban or apixaban in plasma-based andwhole-blood coagulation assays at both therapeutic and supratherapeuticconcentrations of the inhibitor.

The results of these studies were confirmed and extended by testing ifFXa^(I16L) could counteract the anti-coagulant effect of rivaroxabanwhen both agents were administered in vivo. In these experiments,C57BL/6 mice were infused with rivaroxaban (1 mg/kg) or buffer via thetail vein. Mice were then prepared to expose the jugular vein and thevena cava. Approximately 10 min later FXa^(I16L) (1 or 2 mg/kg) wasinfused by direct injection into the jugular vein. Five minutes postinjection blood was collected via the vena cava into citrate and corntrypsin inhibitor. Collected blood was then analyzed by ROTEM usingdilute tissue factor (Innovin, 1:42,000 dilution). Whole blood from miceadministered buffer only clotted by about 2 min (FIG. 12).Administration of 1 mg/kg rivaroxaban substantially prolonged the clottime to about 10 min (FIG. 12). Further administration of FXa^(I16L)shortened clotting time in the presence of rivaroxaban in a dosedependent manner (FIG. 12).

Example 4 FXa^(I16L) Counteracts Rivaroxaban in a Thrombin GenerationAssay

The effect of FXa^(I16L) on reversing rivaroxaban in plasma was examinedin a thrombin generation assay (TGA) using the calibrated automatedthrombography (CAT) system (Thrombinoscope BV, Maastricht, TheNetherlands). Normal human plasma was obtained from George KingBiomedical (Overland Park, Kan.). In the reaction, 20 μL of PPP-ReagentLOW containing 4 μM phospholipids and 1 pM tissue factor was added to 70μL of pooled citrated normal human plasma (treated with 250 nMrivaroxaban, within the therapeutic plasma concentration range) in anImmulon 2HB round bottom 96 well plate with reactions duplicated.Immediately preceding reaction initiation, 10 μL of vehicle orFXa^(I16L) was added to plasma at final concentrations ranging from0.03125 nM to 0.5 nM FXa^(I16L), given a 120 μL total reaction volume.Reactions were initiated by addition of 20 μL FluCa buffer containingcalcium chloride and fluorogenic substrate. Fluorescence of plasmareactions was read at 37° C. at 20 second intervals on a FluoroskanAscent fluorometer and compared to those of reference thrombincalibrator reactions to determine thrombin concentrations. The intensityof the fluorescence signal (FU) was continuously monitored at 37° C.using the CAT. Thrombin generation curves (nM thrombin vs. time) wereanalyzed to extract lag time, peak height, time to peak, and the areaunder the curve representing the endogenous thrombin potential (ETP)using the Thromboscope software (Thrombinoscope BV version).

A dose dependent inhibition of thrombin generation in normal humanplasma was observed with in vitro rivaroxaban treatment (5-200 nM) (FIG.10A). Rivaroxaban resulted in an increase in the lag time coupled with adecrease in the peak thrombin and a decrease in the ETP. The addition ofFXa^(I16L) to rivaroxaban (250 nM) inhibited human plasma resulted in adose dependent reversal of thrombin inhibition (FIG. 10B): peak thrombingeneration was restored, the lag phase was shorter, and the ETPincreased. At a low dose of 0.03125 nM FXa^(I16L), thrombin generationwas restored to levels comparable to vehicle treated normal humanplasma.

Example 5 FXa^(I16L) Counteracts Rivaroxaban in a Mouse Tail ClipBleeding Model

The ability of FXa^(I16L) to overcome the effects of rivaroxaban in vivowas assessed in an acute bleeding model in normal mice. The resultsdemonstrated that a zymogen-like FXa variant could reverse theanticoagulant effect of a direct FXa inhibitor.

To establish a dose of rivaroxaban that would prolong bleeding, maleC57B1/6 mice (The Jackson Laboratory, Bar Harbor, Me.) received a singleintravenous injection of rivaroxaban at a dose of 10, 25 or 50 mg/kg.Thirty minutes later, mice were anesthetized with isoflurane and placedon a heated platform, and the body temperature of the mice wasmaintained at 37° C. prior to the tail cut. The tails were immersed in50 mL pre-warmed phosphate buffered saline (PBS) at 37° C. for 2minutes. A 3 mm tail cut was made and blood was collected into PBS for a10 minute period. A quantitative assessment of the amount of bleedingwas determined by hemoglobin content of the blood collected into PBS.Tubes were centrifuged to collect erythrocytes, resuspended in 5 mLlysis buffer (8.3 g/L NH₄Cl, 1.0 g/L KHCO₃, and 0.037 g/L EDTA), and theabsorbance of the sample was measured at 575 nm. The absorbance valueswere converted to total blood loss (μL) using a standard curve. Theadministration of rivaroxaban resulted in a dose dependent increase inblood loss following a tail cut (FIG. 11).

In this model, a dose of 50 mg/kg rivaroxaban resulted in an increase inblood loss following the tail transection. Mice were dosed with 50 mg/kgrivaroxaban and 30 minutes later 50 or 200 ug/kg of FXa^(I16L) was dosedintravenously at 37° C. prior to the tail cut. Mice were thenanesthetized with isoflurane and placed on a heated platform, and thebody temperature of the mice was maintained at 37° C. prior to the tailcut. The tails were immersed in 50 mLs pre-warmed phosphate bufferedsaline (PBS) at 37° C. for 2 minutes. A 3 mm tail cut was made and bloodwas collected into PBS for a 10 minute period and the assessment of theamount of bleeding was determined by hemoglobin content as described. Inthis model, the administration of the hemostatic FXa^(I16L) variantdecreased the excessive bleeding loss induced with rivaroxaban (FIG.11).

Example 6 FXa^(I16L) Counteracts Rivaroxaban in a Mouse Bleeding ModelDemonstrated Using Intravital Microscopy

As visualized using intravital microscopy, rivaroxaban was demonstratedto inhibit thrombus formation in the microcirculation of the mousecremaster muscle after laser-induced injury. Further administration ofFXa^(I16L) could counteract the anti-coagulant effect of rivaroxaban inthis system.

Using standard techniques, the cremaster muscle of mice was exposed andvisualized using intravital microscopy. A vascular injury in the musclewas then induced using a laser. After injury, clot formation wasvisualized using different fluorescently labeled antibodies thatspecifically recognize fibrin and platelets. Clotting is indicated bythe presence of fluorescent signal from both types of antibodies.

After laser injury, an untreated mouse rapidly formed a clot at the siteof injury that was stable for several minutes (FIG. 13A). In the videoframe, the clot is visible as the coincidence of fluorescent signalassociated with antibodies against fibrin and platelets (light graycenter region overlapping darker gray region). Administration of 1 mg/kgrivaroxaban to a mouse, however, delayed the accumulation of plateletsat the injury site and eliminated any signs of fibrin (FIG. 13B). In thevideo frame, only a reduced extent of platelets can be seen as indicatedby the dark gray region, which reflects presence of fluorescent signalassociated with anti-platelet antibodies. By contrast, when a mouse wasadministered 1 mg/kg rivaroxaban followed by 0.5 mg/kg FXa^(I16L), aclot rapidly formed at the injury site (FIG. 13C). In the video frame,the clot is indicated by the characteristic pattern of fluorescentsignal associated with antibodies against platelets and fibrin.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclature used in connection with, and techniques of, cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003), incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclature used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart.

All publications, patents, patent applications or other documents citedherein are hereby incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, or other document was individually indicated to beincorporated by reference for all purposes.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

What is claimed is:
 1. A method for reducing or preventing bleeding in asubject being treated with a direct Factor Xa inhibitor, comprisingadministering to said subject a Factor Xa variant that contains at leastone modification selected from the group consisting of: a) the aminoacid at the position corresponding to 235 in SEQ ID NO:1 is substitutedwith Thr, Leu, Phe, Asp or Gly; and b) the amino acid at the positioncorresponding to 236 in SEQ ID NO:1 is substituted with Leu, Ala, orGly.
 2. A method for reducing or preventing bleeding in a subject beingtreated with rivaroxaban or apixaban, comprising administering to saidsubject a Factor Xa variant in which the amino acid at the positioncorresponding to 235 in SEQ ID NO:1 is substituted with Leu or Thr.
 3. Apharmaceutical composition for reducing or preventing bleeding in asubject being treated with a direct Factor Xa inhibitor, comprising aFactor Xa variant that that contains at least one modification selectedfrom the group consisting of: a) the amino acid at the positioncorresponding to 235 in SEQ ID NO:1 is substituted with Thr, Leu, Phe,Asp or Gly; and b) the amino acid at the position corresponding to 236in SEQ ID NO:1 is substituted with Leu, Ala, or Gly.
 4. A pharmaceuticalcomposition for reducing or preventing bleeding in a subject beingtreated with rivaroxaban or apixaban, comprising a Factor Xa variant inwhich the amino acid at the position corresponding to 235 in SEQ ID NO:1is substituted with Leu or Thr.
 5. Use of a Factor Xa variant in theproduction of a medicament for reducing or preventing bleeding in asubject being treated with a direct Factor Xa inhibitor, wherein theFactor Xa variant contains at least one modification selected from thegroup consisting of: a) the amino acid at the position corresponding to235 in SEQ ID NO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b)the amino acid at the position corresponding to 236 in SEQ ID NO:1 issubstituted with Leu, Ala, or Gly.
 6. Use of a Factor Xa variant in theproduction of a medicament for reducing or preventing bleeding in asubject being treated with rivaroxaban or apixaban, wherein the aminoacid at the position corresponding to 235 in SEQ ID NO:1 is substitutedwith Leu or Thr.
 7. The method, composition or use of any one of claims1-6, wherein there is a reduction in bleeding of at least about 5%-10%,10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%,50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%,90%-95%, or 95%-100%.
 8. A method for increasing the amount of thrombinproduced in the presence of a direct Factor Xa inhibitor in a subject inneed thereof, comprising administering to said subject a Factor Xavariant that contains at least one modification selected from the groupconsisting of: a) the amino acid at the position corresponding to 235 inSEQ ID NO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) theamino acid at the position corresponding to 236 in SEQ ID NO:1 issubstituted with Leu, Ala, or Gly.
 9. A method for increasing the amountof thrombin produced in the presence of rivaroxaban or apixaban in asubject in need thereof, comprising administering to said subject aFactor Xa variant in which the amino acid at the position correspondingto 235 in SEQ ID NO:1 is substituted with Leu or Thr.
 10. Apharmaceutical composition for increasing the amount of thrombinproduced in the presence of rivaroxaban or apixaban in a subject in needthereof, comprising a Factor Xa variant in which the amino acid at theposition corresponding to 235 in SEQ ID NO:1 is substituted with Leu orThr.
 11. A pharmaceutical composition for increasing the amount ofthrombin produced in the presence of a direct Factor Xa inhibitor in asubject in need thereof, comprising a Factor Xa variant that thatcontains at least one modification selected from the group consistingof: a) the amino acid at the position corresponding to 235 in SEQ IDNO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) the aminoacid at the position corresponding to 236 in SEQ ID NO:1 is substitutedwith Leu, Ala, or Gly.
 12. Use of a Factor Xa variant in the productionof a medicament for increasing the amount of thrombin produced in thepresence of rivaroxaban or apixaban, wherein the Factor Xa variantcontains at least one modification selected from the group consistingof: a) the amino acid at the position corresponding to 235 in SEQ IDNO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) the aminoacid at the position corresponding to 236 in SEQ ID NO:1 is substitutedwith Leu, Ala, or Gly.
 13. Use of a Factor Xa variant in the productionof a medicament for increasing the amount of thrombin produced in thepresence of a direct Factor Xa inhibitor, wherein the amino acid at theposition corresponding to 235 in SEQ ID NO:1 is substituted with Leu orThr.
 14. The method, composition or use of any one of claims 8-13,wherein the amount of thrombin produced increases at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold,25-fold, 30-fold, or 50-fold.
 15. A method for decreasing clotting timein the presence of a direct Factor Xa inhibitor in a subject in needthereof, comprising administering to said subject a Factor Xa variantthat contains at least one modification selected from the groupconsisting of: a) the amino acid at the position corresponding to 235 inSEQ ID NO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) theamino acid at the position corresponding to 236 in SEQ ID NO:1 issubstituted with Leu, Ala, or Gly.
 16. A method for decreasing clottingtime in the presence of rivaroxaban or apixaban in a subject in needthereof, comprising administering to said subject a Factor Xa variant inwhich the amino acid at the position corresponding to 235 in SEQ ID NO:1is substituted with Leu or Thr.
 17. A pharmaceutical composition fordecreasing clotting time in the presence of a direct Factor Xa inhibitorin a subject in need thereof, comprising a Factor Xa variant that thatcontains at least one modification selected from the group consistingof: a) the amino acid at the position corresponding to 235 in SEQ IDNO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) the aminoacid at the position corresponding to 236 in SEQ ID NO:1 is substitutedwith Leu, Ala, or Gly.
 18. A pharmaceutical composition for decreasingclotting time in the presence of rivaroxaban or apixaban in a subject inneed thereof, comprising a Factor Xa variant in which the amino acid atthe position corresponding to 235 in SEQ ID NO:1 is substituted with Leuor Thr.
 19. Use of a Factor Xa variant in the production of a medicamentfor decreasing clotting time in the presence of a direct Factor Xainhibitor in a subject in need thereof, wherein the Factor Xa variantcontains at least one modification selected from the group consistingof: a) the amino acid at the position corresponding to 235 in SEQ IDNO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b) the aminoacid at the position corresponding to 236 in SEQ ID NO:1 is substitutedwith Leu, Ala, or Gly.
 20. Use of a Factor Xa variant in the productionof a medicament for decreasing clotting time in the presence ofrivaroxaban or apixaban in a subject in need thereof, wherein the aminoacid at the position corresponding to 235 in SEQ ID NO:1 is substitutedwith Leu or Thr.
 21. The method, composition or use of any one of claims15-20, wherein there is a reduction in clotting time of at least about5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%,45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%,85%-90%, 90%-95%, or 95%-100%.
 22. The method, composition or use of anyone of claims 15-20, wherein the reduction in clotting time is measuredusing prothrombin time (PT).
 23. The method of claim 22, wherein said PTin said subject is about 25 seconds, 24 seconds, 23 seconds, 22 seconds,21 seconds, 20 seconds, 19 seconds, 18 seconds, 17 seconds, 16 seconds,15 seconds, 14 seconds, 13 seconds, 12 seconds, 11 seconds, or 10seconds.
 24. The method of claim 22, wherein the InternationalNormalized Ratio (INR) in said subject is about 4.0, 3.9, 3.8, 3.7, 3.6,3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2,2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, or0.7.
 25. The method of any one of claim 22, 23, or 24, wherein PT isdetermined 15 mins, 20 mins, 30 mins, 40 mins, 45 mins, 50 mins, 60mins, 75 min, or 90 min after administration of the FXa variant.
 26. Apharmaceutical composition comprising a Factor Xa variant that containsat least one modification selected from the group consisting of: a) theamino acid at the position corresponding to 235 in SEQ ID NO:1 issubstituted with Thr, Leu, Phe, Asp or Gly; and b) the amino acid at theposition corresponding to 236 in SEQ ID NO:1 is substituted with Leu,Ala, or Gly. wherein said Factor Xa variant counters the effect of adirect Factor Xa inhibitor at a plasma concentration of at least100-fold lower than the plasma concentration of the direct Factor Xainhibitor.
 27. A pharmaceutical composition comprising a Factor Xavariant in which the amino acid at the position corresponding to 235 inSEQ ID NO:1 is substituted with Leu or Thr, and wherein the Factor Xavariant counters the effect of rivaroxaban or apixaban at a plasmaconcentration of at least 100-fold lower than the plasma concentrationof the rivaroxaban or apixaban.
 28. The method, composition or use ofany of the previous claims, wherein the Factor Xa variant counters theeffect of a direct Factor Xa inhibitor at a plasma concentration of atleast 100-fold lower than the plasma concentration of the direct FactorXa inhibitor.
 29. The method, composition or use of any of the previousclaims, wherein the Factor Xa variant is administered before a plannedsurgery, after an injury or after a direct Factor Xa inhibitor overdose.30. The method, composition or use of any of the previous claims,wherein Factor Xa variant is administered more than one time.
 31. Themethod, composition or use of any of the previous claims, wherein atleast one additional procoagulant is administered.
 32. The method,composition or use of claim 30, wherein the procoagulant is selectedfrom the group consisting of: a different Factor Xa variant, Factor IX,Factor XIa, Factor XIIa, Factor VIII, Factor VIIa, FEIBA and prothrombincomplex concentrate (PCC).
 33. The method, composition or use of any ofthe previous claims, wherein the plasma concentration of the direct FXainhibitor is a supratherapeutic amount.
 34. The method, composition oruse of any of the previous claims, wherein the direct FXa inhibitor isrivaroxaban and wherein the plasma concentration of rivaroxaban is atleast about 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, or800 nM.
 35. The method, composition or use of any of the previousclaims, wherein the direct FXa inhibitor is apixaban and wherein theplasma concentration of apixaban is at least about 50 nM, 100 nM, 150nM, 200nM, 250 nM, 300 nM, 350 nM, or 400 nM.
 36. A method for effectingthe urgent reversal of acquired coagulopathy due to FXa inhibitiontherapy in a subject with acute major bleeding, comprising administeringto said subject a Factor Xa variant that contains at least onemodification selected from the group consisting of: a) the amino acid atthe position corresponding to 235 in SEQ ID NO:1 is substituted withThr, Leu, Phe, Asp or Gly; and b) the amino acid at the positioncorresponding to 236 in SEQ ID NO:1 is substituted with Leu, Ala, orGly.
 37. A method for effecting the urgent reversal of acquiredcoagulopathy due to FXa inhibition therapy in a subject with acute majorbleeding, comprising administering to said subject a Factor Xa variantin which the amino acid at the position corresponding to 235 in SEQ IDNO:1 is substituted with Leu or Thr.
 38. A pharmaceutical compositionfor effecting the urgent reversal of acquired coagulopathy due to FXainhibition therapy in a subject with acute major bleeding, comprising aFactor Xa variant that that contains at least one modification selectedfrom the group consisting of: a) the amino acid at the positioncorresponding to 235 in SEQ ID NO:1 is substituted with Thr, Leu, Phe,Asp or Gly; and b) the amino acid at the position corresponding to 236in SEQ ID NO:1 is substituted with Leu, Ala, or Gly.
 39. Apharmaceutical composition for effecting the urgent reversal of acquiredcoagulopathy due to FXa inhibition therapy in a subject with acute majorbleeding, comprising a Factor Xa variant in which the amino acid at theposition corresponding to 235 in SEQ ID NO:1 is substituted with Leu orThr.
 40. Use of a Factor Xa variant in the production of a medicamentfor effecting the urgent reversal of acquired coagulopathy due to FXainhibition therapy in a subject with acute major bleeding, wherein theFactor Xa variant contains at least one modification selected from thegroup consisting of: a) the amino acid at the position corresponding to235 in SEQ ID NO:1 is substituted with Thr, Leu, Phe, Asp or Gly; and b)the amino acid at the position corresponding to 236 in SEQ ID NO:1 issubstituted with Leu, Ala, or Gly.
 41. Use of a Factor Xa variant in theproduction of a medicament for effecting the urgent reversal of acquiredcoagulopathy due to FXa inhibition therapy in a subject with acute majorbleeding, wherein the amino acid at the position corresponding to 235 inSEQ ID NO:1 is substituted with Leu or Thr.